Mitochondria code for only a limited number of constituent proteins. The majority of mitochondrial proteins are encoded in the nucleus and synthesized in the cytoplasm. One of the fundamental questions related to mitochondrial biogenesis is how cytoplasmically synthesized proteins cross the existing outer and inner membranes and are incorporated into the mitochondrion. To clarify this queston, the biosynthesis of carbamyl phosphate synthetase and ornithine transcarbamylase, two enzymes of the mitochondrial matrix is being investigated. These enzymes are synthesized on cytoplasmic ribosomes in the form of precursors. Each enzyme has been shown to be some 5000 daltons larger than the mature enzyme. In order to derive the amino acid sequence of the precursor as compared to the native enzyme, the carbamyl phosphate synthetase gene from rat will be cloned into E. coli, and sequenced. The cloned DNA will also be used as a probe to study the regulation of synthesis of the enzyme.